Dairy Propionibacteria As Human Probiotics: a Review of Recent Evidence Fabien J

Dairy propionibacteria as human probiotics: A review of recent evidence Fabien J. Cousin, Denis D G Mater, Benoît Foligné, Gwénaël Jan

To cite this version:

Fabien J. Cousin, Denis D G Mater, Benoît Foligné, Gwénaël Jan. Dairy propionibacteria as human probiotics: A review of recent evidence. Dairy Science & Technology, EDP sciences/Springer, 2011, 91 (1), pp.1-26. 10.1051/dst/2010032. hal-00868601

HAL Id: hal-00868601 https://hal.archives-ouvertes.fr/hal-00868601 Submitted on 16 Oct 2013

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Dairy propionibacteria as human probiotics: A review of recent evidence

Fabien J. Cousin & Denis D. G. Mater & Benoît Foligné & Gwénaël Jan

Received: 3 November 2009 /Revised: 29 April 2010 /Accepted: 29 April 2010 / Published online: 2 August 2010 # INRA, EDP Sciences, 2010

Abstract Probiotics have been the subject of intensive research, mainly focusing on bifidobacteria and lactic acid bacteria. However, there is evidence that dairy propionibacteria also display probiotic properties, which as yet have been under- estimated. The aim of this paper is to review recent data which report probiotic characteristics of dairy propionibacteria and to distinctly organise them based on the experimental strategy employed: ranked from in vitro evidence to in vivo trials, which is a new approach. In addition to the selection criteria for probiotics in areas such as food safety, technological and digestive stress tolerance, many potential health benefits have been described which include modulation of microbiota and metabolic activity in the gut, modulation of intestinal motility and absorption, impact on intestinal inflammation, modulation of the immune system and potential modulation of risk factors for cancer development. The robust nature of dairy propionibacteria towards technological stresses should allow their future use in various fermented probiotic foods. Among the probiotic properties of dairy propionibacteria described in the literature, some of these properties are different from those reported for bifidobacteria and lactic acid bacteria. However, supplementation with dairy propionibacteria in randomised, placebo-controlled, double-

This article should be cited as: Cousin F.J. et al., Dairy propionibacteria as human probiotics: A review of recent evidence, Dairy Science & Technology (2010), doi:10.1051/dst/2010032 F. J. Cousin : G. Jan (*) INRA, UMR1253 Science et Technologie du Lait et de l’Oeuf, 35042 Rennes, France e-mail: [email protected]

F. J. Cousin : G. Jan AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l’Oeuf, 35042 Rennes, France

F. J. Cousin : D. D. G. Mater CNIEL/Syndifrais, 42 rue de Châteaudun, 75314 Paris 09, France

B. Foligné Institut Pasteur de Lille, Bactéries Lactiques & Immunité des Muqueuses, Centre d’Infection et d’Immunité de Lille, Unit 1019—UMR 8204, Univ. Lille Nord de France, 59019 Lille, France 2 F.J. Cousin et al. blind human trials has mainly involved mixtures of propionibacteria with probiotic bacteria from other genera. Clinical studies involving the use of dairy propionibacteria alone are lacking. Such studies will allow the specifically observed health benefits to be attributed to dairy propionibacteria. This, in turn, will allow the investigation of the synergistic effects with other probiotic bacteria or beneficial food components.

摘要益生性丙酸菌的研究进展。近年来关于双歧杆菌和乳酸菌的益生性得到了广泛的关注、相关的研究报道较多。尽管乳中的丙酸菌也显示具有益生菌的功能、但并没有引起人们的重视。本文综述了近年来关于乳源丙酸菌益生功能的研究进展。论述的内容包括从食品安全性考虑益生菌的选择标准、益生菌的消化极限; 以及益生菌潜在的功能性、如对肠道中微生物菌群和代谢活性的调整作用、促进肠道蠕动和吸收作用、对肠炎的作用、对免疫系统的调节作用以及潜在的对癌症因子抑制作用。丙酸菌在乳中生产旺盛、因此从技术层面上分析丙酸菌可以用于各种发酵食品。在关于乳源丙酸菌益生功能性的文献报道中、丙酸菌的一些功能性不同于双歧杆菌和乳酸菌。然而、在双目失明的人体试验中、则是将来源于其他属的丙酸菌与乳源丙酸菌混合使用。在临床试验中通常用含丙酸菌的乳制品和不含丙酸菌的乳制品同时进行试验、目的是证明乳源丙酸菌对人体健康的作用。同样方法也可以调查丙酸菌与其他益生菌或食物中其他有益成分的协同作用。

Keywords Probiotic . Propionibacteria . Propionibacterium . Short-chain fatty acid

关键词益生性 . 丙酸菌 . 丙酸杆菌属 . 短链脂肪酸

Abbreviations ACNQ 2-amino-3-carboxy-1,4- naphthoquinone AEDS Atopic eczema-dermatitis syndrome BGS Bifidogenic growth stimulator CLA Conjugated linoleic acid CMA Cow’s milk allergy CRP C-reactive protein DHNA 1,4-dihydroxy-2-naphthoic acid DMH 1,2-dimethylhydrazine DSS Dextran sodium sulphate EFSA European food safety authority GRAS Generally recognised as safe IBD Inflammatory bowel diseases IBS Irritable bowel syndrome NSAID Nonsteroidal anti-inflammatory drug QPS Qualified presumption of safety SCFA Short-chain fatty acid TNBS Trinitrobenzene sulphonic acid

1 Introduction

Propionibacteria were first described at the end of the 19th century by E. von Freudenreich and S. Orla-Jensen, studying propionic acid fermentation in Emmental Dairy propionibacteria as probiotics 3 cheese, leading to propose the genus Propionibacterium (Orla-Jensen 1909). Propioni- bacteria are firmicutes with a high G+C content, characterised as gram-positive, non- sporing, non-motile pleomorphic rods. They are anaerobic to aerotolerant and generally catalase positive. They grow optimally at 30 °C and are of neutral pH. Cells are hetero- fermentative and metabolise a variety of substrates such as carbohydrates (including glucose, galactose, fructose and lactose), alcohols (glycerol and erythritol) and organic acids (lactate and pyruvate). Propionibacteria present a particular central carbon metabolic pathway, propionic fermentation. This fermentation involves the Wood- Werkmann cycle (Wood et al. 1981) which produces propionate, acetate, succinate and carbon dioxide. The genus Propionibacterium is divided in two groups (Table 1) based on habitat of origin: classical or dairy propionibacteria (mainly isolated from dairy products such as cheese) and cutaneous propionibacteria (typically found on skin). Recently, a new species, Propionibacterium acidifaciens, was isolated from human carious dentine (Downes and Wade 2009). It was proposed as a member of the cutaneous group because it is closely related to Propionibacterium australiense. Dairy propionibacteria are commonly used as starter cultures in the dairy industry. Their main application is the ripening of Swiss-type cheeses, characterised by round “eyes”. In these cheeses, they play an important role in characteristic flavour and opening. Opening is due to the production of carbon dioxide. Flavour is linked to the production by dairy propionibacteria of volatiles, mainly propionate and acetate, and other compounds derived from amino acid and lipid catabolisms (Dherbecourt et al. 2008, 2010; Langsrud and Reinbold 1973; Thierry et al. 2002, 2004a, b). Some strains of dairy propionibacteria are also used in probiotic preparations, alone or in combination with lactic acid bacteria and/or bifidobacteria. A probiotic is defined as “a live microorganism which, when administered in adequate amounts, confers a health benefit on the host” (FAO et al. 2006). An increasing number of reports on potential probiotic properties of propionibacteria have been published. However, there is far less literature on the probiotic properties of propionibacteria than on the lactobacilli and bifidobacteria. Two book chapters (Jan et al. 2007; Ouwehand et al. 2004) and two earlier reviews (Mantere-Alhonen 1995; Vorobjeva et al. 1995) have reported on these potentialities. Recent evidence has been described in the literature but never reviewed. Moreover, no one has clearly sorted the probiotic characteristics

Table 1 Dairy and cutaneous Propionibacterium species

Dairy (classical) propionibacteria Cutaneous propionibacteria

P. acidipropionici P. acidifaciens P. cyclohexanicum P. acnes P. freudenreichii subsp. freudenreichii P. australiense P. freudenreichii subsp. shermanii P. avidum P. jensenii P. granulosum P. microaerophilum P. propionicum P. thoenii

The species formerly known as P. inoccuum and P. lymphophilum have been reclassified as Propioniferax innocua (Yokota et al. 1994) and Propionimicrobium lymphophilum (Stackebrandt et al. 2002), respectively. 4 F.J. Cousin et al. described according to the level of evidence in the reports (in vitro, ex vivo, in vivo in animal models and in human beings). In addition, clinical studies have mainly reported on the use of dairy propionibacteria in complex bacterial mixtures and rarely with propionibacteria alone. This review provides an update on the probiotic potentialities of dairy propionibacteria and organises the potential health benefits according to the scientific level of the evidence. The paper only covers dairy propionibacteria, and only their probiotic potentialities in the gut. Uses as food preservatives and animal probiotics are not described here but have been extensively discussed in reports mentioned above. This paper includes six sections reviewing first selection criteria for using propionibacteria as probiotics, then probiotic potentialities of dairy propionibacteria that are suggested distinctly in vitro, ex vivo, in vivo in animal models and in human clinical study. In each section, we will successively evoke the impact of dairy propionibacteria on the gut microbiota (lowering pathogenic microorganisms and favouring beneficial ones), on different aspects of gut mucosa physiology and on the immune system. Finally, the production of others compounds related to propionibacteria and future trends in their further applications are discussed.

2 Selection criteria for using propionibacteria as probiotics

Selection of bacteria for probiotic application relies on criteria such as safety, technological and digestive stress survival, intestinal cell adhesion and human origin. The two last conditions are controversial and it is now recognised that they are not mandatory, although in some cases they may improve probiotic potential (Sanders 2008). These selection criteria are summarised in Table 2. The safety of dairy propionibacteria is evidenced by the widespread consumption of Swiss-type cheese. The highest rate of Emmental consumption is found in France, where per capita consumption averages 4 kg·year−1. Propionibacteria are present in Emmental in concentrations close to 109 bacteria per gram, and species Propionibacterium freudenreichii received the “Generally Recognised As Safe” (GRAS) (Mogensen et al. 2002). This opens the way to other applications such as probiotics, as discussed below. For example, one criterion studied for probiotic use is the absence of antibiotic resistance, because of the risk that any resistance will spread to intestinal microbiota. Dairy propionibacteria have natural resistance to few antibiotics and this resistance does not appear to be encoded by plasmids or other mobile genetic elements (Meile et al. 2008). In addition, dairy propionibacteria do not possess any known virulence factors, although some Propionibacterium thoenii and Propionibacterium jensenii strains have β-haemolytic activity (Meile et al. 2008). The European food safety authority has granted “Qualified presumption of safety” (QPS) status to species P. freudenreichii (Anonymous et al. 2008). No cytotoxic effect in mouse colonocytes (Zarate 2009) and no effect on the general health of rats (Huang et al. 2003; Lan et al. 2007a, 2008) or infants (Kukkonen et al. 2008) have been observed with strains of dairy propionibacteria. No indication of side effects from consuming dairy propionibacteria has been reported in any of the human trials reviewed here (Bouglé et al. 1999; El-Nezami et al. 2000b, 2006; Hatakka et al. 2007, 2008; Hervé et al. 2007; Hojo et al. 2002; Jan et al. 2002b; Dairy propionibacteria as probiotics 5

Kajander et al. 2005, 2007, 2008; Kajander and Korpela 2006; Kaneko 1999; Kekkonen et al. 2008b; Kuitunen et al. 2009; Kukkonen et al. 2007, 2008; Mitsuyama et al. 2007; Myllyluoma et al. 2005, 2007a, b; Ouwehand et al. 2004; Roland et al. 1998; Sarkar and Misra 1998, 2002; Satomi et al. 1999; Seki et al. 2004; Sidorchuk and Bondarenko 1984; Suzuki et al. 2006; Viljanen et al. 2005a, b). In addition to their tolerance to technological stresses (cheese making, encapsulation, freeze-drying etc.), dairy propionibacteria present good constitutive survival under digestive stress. In vitro, many studies have described their natural ability to survive low pH conditions and exposure to bile (Huang and Adams 2004; Jan et al. 2002b; Leverrier et al. 2005; Mantere-Alhonen 1983; Suomalainen et al. 2008; Warminska-Radyko et al. 2002; Zarate et al. 2000). This tolerance is reinforced by a brief exposure to the same stress at a non-lethal level (Jan et al. 2000, 2001; Leverrier et al. 2003, 2004). Propionibacteria adapted in this way are able to survive pH values as low as two and bile salts’ concentrations higher than those reported in the human gut. The probiotic vector is also important for digestive stress tolerance. Propionibacteria in cheese had better tolerance to acid challenge than free cultures (Jan et al. 2000). A yoghurt-type fermented milk provided P. freudenreichii with a high tolerance towards acid challenge, bile salts challenge and a succession of the two (Leverrier et al. 2005). Survival during gastrointestinal transit has also been reported in vivo in rodents (Huang et al. 2003; Lan et al. 2007a; Perez-Chaia and Zarate 2005) and human beings (Bouglé et al. 1999; Jan et al. 2002b; Suomalainen et al. 2008). A very high level of propionibacteria was detected in faeces but this concentration returned to the initial level a few days (orweeks) after consumption ceased. Although controversial, another reported criterion for selecting probiotics is the ability to adhere to intestinal cells and/or mucosa. Dairy propionibacteria are able to adhere to immobilised mucus (Ouwehand et al. 2000; Thiel et al. 2004; Tuomola et al. 1999) and this adhesion was increased by the presence of other probiotic bacteria (Collado et al. 2007a; Ouwehand et al. 2002b). The authors suggest that adhesion to mucus is the result of non-specific interactions, because adhesion to mucus and to bovine serum albumin was similar. Dairy propionibacteria also adhere in vitro to human intestinal epithelial cell lines (Huang and Adams 2003; Lehto and Salminen 1997; Moussavi and Adams 2010). Zarate et al. (2002b) demonstrated the adhesion of propionibacteria ex vivo to isolated mouse intestinal epithelial cells and in vivo by a plate count of the viable propionibacteria adhering to intestinal cells. These properties of dairy propionibacteria–safety, gastrointestinal transit survival and adherence to intestinal cells and mucosa–offer good prospects for their use as human probiotics. Table 3 summarises promising properties further described below.

3 Probiotic potentialities of dairy propionibacteria suggested in vitro

As regards modulation of gut microbiota, some authors have described a reduction in pathogen adhesion to immobilised mucus in the presence of P. freudenreichii subsp. shermanii JS, alone or in combination with other probiotic bacteria (Collado et al. 2006, 2007b, c, 2008; Myllyluoma et al. 2008). Collado et al. (2008) showed that P. freudenreichii subsp. shermanii JS is able to aggregate with pathogenic bacteria. 6 Table 2 Selection criteria for using dairy propionibacteria as probiotics

Characteristic/effect Described in vitro Described ex vivo Described in animals Described in humans

Safety N.D. No decrease in viability of No effect on the general health GRAS and QPS status (Meile et al. 2008; mouse colonocytes with of rats (Huang et al. 2003; Lan Mogensen et al. 2002). Emmental P. freudenreichii and et al. 2007a, 2008). consumption. No effect on the general P. acidipropionici health in human beings (Kukkonen et al. (Zarate 2009). 2008; Suzuki et al. 2006). Digestive stress Constitutive acid and bile tolerance N.D. High propionibacteria survival in P. freudenreichii survival in human beings tolerance and (Huang and Adams 2004; Jan et al. rats (Huang et al. 2003; Lan et al. (Bouglé et al. 1999; Jan et al. 2002b; survival in the gut 2002b; Leverrier et al. 2005; Mantere- 2007a) and mice (Perez-Chaia and Suomalainen et al. 2008). 5E+10 − − Alhonen 1983; Suomalainen et al. 2008; Zarate 2005). 1–5E+10 cfu·day 1a cfu·day 1a (Bouglé et al. 1999). 5E+09 − − Warminska-Radyko et al. 2002; Zarate (Huang et al. 2003). 2E+0 cfu·day 1a and 5E+10 cfu·day 1a (Jan et al. 2002b). − − et al. 2000). Efficient adaptive response (Lan et al. 2007a). 1E+09 cfu·day 1a,b 4–8E+10 cfu·day 1b (Suomalainen to acid (Jan et al. 2000, 2001) and bile (Perez-Chaia and Zarate 2005). et al. 2008). (Leverrier et al. 2003) in P. freudenreichii. Gut adhesion Adhesion of P. freudenreichii to immobilised Adhesion of propionibacteria to Adhesion of propionibacteriaa to mice N.D. mucus (Ouwehand et al. 2000; Thiel et al. isolated mice intestinal cells ileal epithelium (Zarate et al. 2002b). − 2004; Tuomola et al. 1999). Adhesion of (Zarate et al. 2002b). Water with 1E+09 cfu·mL 1. propionibacteria to cultured human intestinal cells (Huang and Adams 2003; Lehto and Salminen 1997; Moussavi and Adams 2010). Increased adhesion of P. freudenreichii to immobilised mucus by probiotics combination (Collado et al. 2007a; Ouwehand et al. 2002b).

N.D.: not determined. a

Propionibacteria alone. al. et Cousin F.J. b Propionibacteria in combination with other probiotic bacteria. Ingested quantities of propionibacteria are given when available. ar rpoiatraa probiotics as propionibacteria Dairy Table 3 Useful characteristics for probiotic applications and beneficial effects reported for dairy propionibacteria

Characteristic/effect Described in vitro Described ex vivo Described in animals Described in humans

Gut microbiota 1) Identification of bifidogenic N.D. 1) Modulation of mice microbiota 1) Enhancement of bifidobacteria by P. compounds (DHNA and ACNQ) in by P. acidipropionicia (Perez- freudenreichiia (Bouglé et al. 1999; Hojo et P. freudenreichii (Isawa et al. 2002; Chaia et al. 1999). Water with al. 2002; Kaneko 1999; Roland et al. 1998; − − Kaneko 1999; Kaneko et al. 1994; 1E+08 cfu·mL 1. Satomi et al. 1999). 5E+10 cfu·day 1 Kouya et al. 2007; Mori et al. 1997; (Bouglé et al. 1999; Roland et al. 1998). Roland et al. 1998; Warminska- Radyko et al. 2002). 2) Coaggregation with pathogen 2) Reduction in the level of 2) Modulation of intestinal microbiota in strains by P. freudenreichii (Collado coliforms and increase in the children by P. freudenreichiib (Sarkar and et al. 2008). level of bifidobacteria and Misra 1998, 2002; Sidorchuk and − lactobacilli in rat faecal Bondarenko 1984). 1E+10 cfu·day 1 microbiota by P. freudenreichiib (Sarkar and Misra 1998). (Sarkar and Misra 1998; 2002). 3) Reduction in pathogens adhesion to 3) Decrease in tissue colonisation 3) Increase in bifidobacteria and decrease in immobilised mucus by P. and increase in time survival to bacteroides in human beings receiving freudenreichii (Collado et al. 2006; S. typhimurium in mice by P. DHNA (Mitsuyama et al. 2007; Seki et al. Collado et al. 2007b, c; acidipropionicia (Alvarez et al. 2004). − Vesterlund et al. 2006). 1996). 1.2E+09 cfu·day 1. 4) Reduction in H. pylori adhesion on 4) Increase in bifidobacteria in 4) Improvement of the tolerance to the intestinal cells by P. freudenreichii mice receiving DHNA (Okada treatment of H. pylori infection by P. (Myllyluoma et al. 2008). et al. 2006a). freudenreichiib (Myllyluoma et al. 2005, − 2007a). 5–9E+10 cfu·day 1 (Myllyluoma et al. 2005). 5) Inhibition of H. pylori-induced cell 5) DHNA recovery of loss of 5) Beneficial effect on gastric mucosa in H. membrane leakage in Caco-2 cells Lactobacillus and pylori infected patients by P. freudenreichiib by P. freudenreichii (Myllyluoma et Enterobacteriaceae by DSS- (Myllyluoma et al. 2007b). 2.5E+ − al. 2008). induced colitis in mice (Okada 09 cfu·day 1. et al. 2006b). 6) Decreased prevalence in oral Candida in elderly by P. freudenreichiib (Hatakka et al. − 2007). 5E+08 cfu·day 1. Beneficial metabolic β-galactosidase activity enhanced in N.D. 1) Increase in β-galactosidase 1) Modulation of SCFA in human faeces by P. activities in the gut the presence of bile by P. activity and propionic acid in freudenreichiia (Jan et al. 2002b). 5E+09 7 8 Table 3 (continued)

Characteristic/effect Described in vitro Described ex vivo Described in animals Described in humans

− acidipropionici (Zarate et al. 2000, mice caecum by P. acidipro- and 5E+10 cfu·day 1. 2002a). pionicia (Perez-Chaia and − Zarate 2005). 1E+09 cfu·day 1. 2) Propionic acid fermentation of 2) Propionic acid fermentation of P. P. freudenreichiia in rats (Lan et freudenreichii in humana (Hervé et al. 2007). − − al. 2007a). 2E+10 cfu·day 1. 1E+11 cfu·day 1. Modulation of Cholesterol uptake by P. Enhanced iron absorption from Decrease in serum cholesterol, in 1) Constipation alleviation by P. intestinal motility freudenreichii (Somkuti and the rat colon in the presence of mice fed with a lipid rich diet, freudenreichiia (Hojo et al. 2002). a and absorption Johnson 1990). P. freudenreichii (Bouglé et al. by P. acidipropionici (Perez- 2) Modulation of digestive motility by 2002). Chaia et al. 1995). Water with a − propionibacteria (Bouglé et al. 1999). 5E+ 1 − 1E+08 cfu·mL . 10 cfu·day 1. 3) Increase in defecation frequency in elderly by P. freudenreichiia,b (Ouwehand et al. 2002a; Seki et al. 2004). Potential modulation 1) Binding of carcinogenic toxins (El- Binding of carcinogenic toxins 1) Decrease in β-glucuronidase 1) Reduction in the faecal level of azoreductase of risk factors for Nezami et al. 1998, 2002; Gratz et (El-Nezami et al. 2000a; Gratz activity in mice caecuma (Perez- activity in elderly subjects by P. cancer al. 2004, 2005; Halttunen et al. et al. 2003, 2005; Zarate 2009). Chaia et al. 1999). Water with freudenreichiib (Ouwehand et al. 2002a). − development 2008; Haskard et al. 2001; Lee et al. 1E+08 cfu·mL 1. 2003; Niderkorn et al. 2006; Zarate 2009). 2) Binding of heavy metals (Halttunen 2) Increase in apoptosis in rats 2) Decrease in β-glucosidase and urease by P. et al. 2008; Ibrahim et al. 2006). treated with DMH by P. freudenreichiib (Hatakka et al. 2008). 2E+ − freudenreichiia (Lan et al. 10 cfu·day 1. −1 3) Antimutagenic properties 2008). 2E+10 cfu·day . 3) Decrease in β-glucuronidase activity by P. (Vorobjeva et al. 1995, 2001, freudenreichiib (Kajander et al. 2007). 2E+ − 2004, 2008) 09 cfu·day 1. ..Cui tal. et Cousin F.J. 4) Induction of apoptosis (Jan et al. 4) Reduction in the faecal level of aflatoxin B1 2002a; Lan et al. 2007b). by P. freudenreichiib (El-Nezami et al. 2000b). 5) Induction of NKG2D ligand 5) Reduction of colon exposure to aflatoxin B1 ar rpoiatraa probiotics as propionibacteria Dairy expression on cancer cells by P. by P. freudenreichiib (El-Nezami et al. − freudenreichii and P. acidipropionici 2006). 2–5E+10 cfu·day 1. (Andresen et al. 2009). Healing on intestinal N.D. N.D. 1) Healing by P. freudenreichiia 1) Improvement of the clinical activity index inflammation and P. acidipropionicia of scores in active ulcerative colitis patients TNBSinduced colitis in rats receiving DHNA (Mitsuyama et al. 2007; (Michel et al. 2005; Uchida and Suzuki et al. 2006). Mogami 2005). 2) Attenuation of DSS-induced 2) Alleviation of the symptoms of irritable colitis in mice by DHNA bowel syndrome by P. freudenreichiib (Okada et al. 2006b). (Kajander et al. 2005, 2008; Kajander and − Korpela 2006). 1–2E+09 cfu·day 1. Immunomodulation 1) Inhibition of H. pylori-induced IL- N.D. 1) Stimulation of mice 1) Decrease in serum levels of CRP by P. a 8 and PGE2 released in Caco-2 cells phagocytosis by P. freudenreichii (Kekkonen et al. 2008b). − by P. freudenreichii (Myllyluoma et acidipropionicia (Morata de 3.3E+10 cfu·day 1. al. 2008). Ambrosini et al. 1998; Perez- Chaia et al. 1995). Water with − 1E+08 cfu·mL 1 (Perez-Chaia et al. 1995). 2) Induction of NKG2D ligand 2) Stimulation of lymphocyte 2) Induction of IL-4 secretion in infants PBMC expression on human-activated T proliferation in mice by P. with CMA by P. freudenreichii (Pohjavuori − lymphocytes by P. freudenreichii freudenreichii and P. jensenii et al. 2004). 2E+09 cfu·day 1. and P. acidipropionici (Andresen et (Adams et al. 2005; Kirjavainen − al. 2009). et al. 1999). 1E+08 cfu·day 1 (Adams et al. 2005). 1E+09 − and 1E+12 cfu·kg 1 body − weight·day 1 (Kirjavainen et al. 1999). 3) Induction of TNF-α and IL-10 in 3) Increase in the level of secreted 3) Prevention of IgE-associated allergy in PBMC by P. freudenreichii (Kek- IgA by P. acidipropionici in caesarean-delivered children by P. freuden- konen et al. 2008a). micea (Alvarez et al. 1996). reichiib (Kuitunen et al. 2009). 2E+ − − x1.2E+09 cfu·day 1. 09 cfu·day 1. 4) Anti-inflammatory effect of 4) Increase in the resistance to respiratory DHNA (Okada et al. 2006a). infections during the first 2 years of life by P. freudenreichiib (Kukkonen et al. 2008). 2E+ − 09 cfu·day 1. 5) Prevention of atopic eczema/dermatis 9 10 Table 3 (continued)

Characteristic/effect Described in vitro Described ex vivo Described in animals Described in humans

syndrome in infants by P. freudenreichiib (Kukkonen et al. 2007; Viljanen et al. − 2005a, b). 4E+09 cfu·day 1.

N.D.: not determined. a Propionibacteria alone. b Propionibacteria in combination with other probiotic bacteria. Ingested quantities of propionibacteria are given when available. ..Cui tal. et Cousin F.J. Dairy propionibacteria as probiotics 11

Myllyluoma et al. (2008) described inhibition of Helicobacter pylori adhesion to a human intestinal epithelial cell line by P. freudenreichii subsp. shermanii JS alone. The authors observed the same inhibition with a combination with other probiotic bacteria. This combination also inhibited H. pylori-induced cell membrane leakage (Myllyluoma et al. 2008). A major advantage of dairy propionibacteria for microbiota modulation is their ability to enhance the growth of bifidobacteria (Kaneko et al. 1994; Moussavi and Adams 2010; Roland et al. 1998; Warminska- Radyko et al. 2002). Two bifidogenic compounds have been already identified in vitro: 1,4-dihydroxy-2-naphthoic acid (DHNA) (Isawa et al. 2002) and 2-amino-3- carboxy-1, 4-naphthoquinone (ACNQ) (Kaneko 1999; Mori et al. 1997). DHNA is a precursor of menaquinone (vitamin K2) biosynthesis in bacteria. Some DHNA is released from propionibacteria during growth (Furuichi et al. 2006). ACNQ stimulated the growth of bifidobacteria at an extremely low concentration (0.5 nM) and enhanced the activity of NADH peroxidase and NADH oxidase in bifidobacteria. ACNQ, which may derive from DHNA, is an electron acceptor of NAD(P)H diaphorase and an electron donor of NAD(P)H peroxidase in bifidobacteria (Kaneko 1999; Mitsuyama et al. 2007; Yamazaki et al. 1998, 1999). NAD(P)+regeneration is thought to be responsible for the ability of propionibacteria to stimulate bifidobacteria growth via DHNA and ACNQ. It is also worth noting that propionate, the main end-product of propionibacteria fermentation, is considered to favour the growth of bifidobacteria (Kaneko et al. 1994); a property used in bifidobacteria selective culture media (Hartemink and Rombouts 1999). As regards modulation of gut enzyme activity, some bacterial species including yoghurt starters are efficient in treating lactose intolerance by enhancing β- galactosidase activity in the intestine (de Vrese et al. 2001). Propionibacterium acidipropionici and P. freudenreichii have a high level of β-galactosidase activity at 37 °C (Zarate et al. 2002a) which was even improved in the presence of bile (Zarate et al. 2000, 2002a), by permeabilisation of cells. The authors conclude that the environment in the human intestine may be adequate for β-galactosidase synthesis and activity. They also suggested that a fermented dairy product constitutes a good vector for high β-galactosidase activity. Moreover, the enzyme withstood the cooking temperature of Swiss-type cheeses and proved to be stable during storage at low temperatures. Propionate was recently shown to enhance expression of epithelial calcium channel ECaC2, which is involved in the transcellular route of intestinal calcium absorption in Caco-2 cells (Fukushima et al. 2009). This would at least partly explain why propionate was shown to enhance calcium absorption from the human colon (Trinidad et al. 1999). Intestinal absorption of lipids may be modulated by the consumption of probiotics, including dairy propionibacteria. Somkuti and Johnson found evidence of adsorption of cholesterol to P. freudenreichii cells. This adsorption was mainly passive, as 70% of the cholesterol removed from the medium could be recovered by solvent extraction fromwashed cells (Somkuti and Johnson 1990). This suggests, however, that the presence of dairy propionibacteria in the gut may reduce the bioavailability of cholesterol. Propionibacteria may contribute to reduction of risk factors for cancer development particularly through the ability to bind, in vitro, carcinogenic compounds like mycotoxins (El-Nezami et al. 2002; Niderkorn et al. 2006) and 12 F.J. Cousin et al.

especially aflatoxin B1 (El-Nezami et al. 2000a; Gratz et al. 2004, 2005; Halttunen et al. 2008; Haskard et al. 2001; Lee et al. 2003), cyanotoxins such as microcystin-LR (Halttunen et al. 2008), plant lectins such as concanavilin A and jacalin (Zarate 2009), and also some heavy metals such as cadmium and lead (Halttunen et al. 2008; Ibrahim et al. 2006). The organic toxins cited above are particularly associated with colorectal cancer.Heavymetals havemany deleterious effects on human health including kidney or other cancers. These data suggest thatdairypropionibacteriamayhelp to reduce gut absorption of carcinogenic compounds and so limit the emergence or development of cancer. In addition, these bacteria may be used as detoxifying additives in food contaminated by high levels of this kind of carcinogenic compound which are very difficult to remove. Vorobjeva et al. (1995, 2001, 2008) demonstrated the antimutagenic properties of dairy propionibacteria. P. freudenreichii subsp. shermanii prevented mutations caused by various mutagenic agents. The suggested active component is a cysteine synthase of 35 kg·mol−1, secreted into the extracellular environment (Vorobjeva et al. 2008). Another potential anti-cancer property is the ability of P. freudenreichii and P. acidipropionici to induce apoptosis of colorectal carcinoma cells owing to the action of shortchain fatty acids, especially propionate, on cancer cell mitochondria (Jan et al. 2002a, Lan et al. 2007b). These data suggest that the use of dairy propionibacteria could reduce the incidence of colon cancer or help to treat this cancer, which is the second most fatal cancer in Europe. In addition, P. freudenreichii and P. acidipropionici induced NKG2D ligand expression on various cancer cells and the authors speculate that the pro-apoptotic effectmay also bemediated by this overexpression (Andresen et al. 2009). P. freudenreichii has been observed to modulate the immune system in vitro by inhibition of H. pylori-induced IL-8 and PGE2 release in human intestinal epithelial cells (Myllyluoma et al. 2008). These anti-inflammatory effects did not persist when P. freudenreichii subsp. shermanii JS was used in combination with Lactobacillus rhamnosus GG, L. rhamnosus LC-705 and B. breve Bbi99. The authors stress the importance of characterising the individual strains to improve the therapeutic response of probiotics used in combination. In addition, P. freudenreichii and P. acidipropionici induced NKG2D ligand MICA/B expression on human-activated T lymphocytes without affecting this expression on resting peripheral blood cells. This effect was also observed with propionate alone and to a lesser extent by acetate (Andresen et al. 2009). Expression is stimulated by higher promoter activity due to a mechanism that depends on intracellular calcium. The authors suggest that preoperative treatment with cutaneous propionibacteria strains may produce beneficial immunostimulation and increased survival of patients with colorectal carcinoma. As regards cytokine production, P. freudenreichii subsp. shermanii JS was able to induce TNF-α and IL- 10 production in human PBMCs (Kekkonen et al. 2008a). The anti-inflammatory actions of IL-10 could be helpful in the treatment of inflammatory conditions or diseases. Interestingly, P. freudenreichii subsp. shermanii JS induced the expression of IL-12 (pro-inflammatory cytokine) only weakly (Kekkonen et al. 2008a), suggesting useful implication to treat colitis as reported earlier (Foligné et al. 2007). E. coli DH5α-induced IFN-γ production was also reducedwhen it was combined with P. freudenreichii subsp. shermanii JS (Kekkonen et al. 2008a). Dairy propionibacteria as probiotics 13

4 Probiotic potentialities of dairy propionibacteria studied ex vivo

P. freudenreichii enhanced iron absorption from the rat proximal colon ex vivo, via the production of short-chain fatty acids (SCFA), especially propionate (Bouglé et al. 2002). The authors suggest that this absorption may be enhanced in vivo by local production of SCFA. This suggests a positive effect of dairy propionibacteria on the bioavailability of dietary iron for uptake by the liver and spleen. The ability of dairy propionibacteria to bind carcinogenic compounds like aflatoxin B1 has also been described ex vivo (El-Nezami et al. 2000a; Gratz et al. 2003, 2005). A mixture of P. freudenreichii subsp. shermanii JS and L. rhamnosus LC- 705 was able to bind aflatoxin B1 and tissue uptake of this carcinogen was reduced when probiotic bacteria were present in a duodenal loop. Consequently, this probiotic mixture could delay, but not prevent, aflatoxin B1 absorption in duodenal loops. Hence, ingestion of dairy propionibacteria may limit bioavailability, absorption and metabolisation of these carcinogenic compounds and so decrease cancer emergence risk.

5 Probiotic potentialities of dairy propionibacteria in animal models

Sarkar and Misra (1998, 2002) showed a decline in the total number of faecal bacteria in rats fed a fermented milk containing Bifidobacterium bifidum, P. freudenreichii subsp. shermanii and either L. acidophilus or not. Especially, the number of coliforms was decreased, but an increase in the bifidobacteria population was observed. Perez-Chaia et al. (1999) noticed a similar microbiota modulation in mice consuming P. acidipropionici, with fewer anaerobes and coliforms in the caecal content. Moreover, Alvarez et al. (1996) reported that feeding a P. acidipropionici CRL 1198 supplement to mice prior to Salmonella typhimurium administration afforded a partial protection against the pathogen colonisation. Indeed, a decrease in tissue colonisation by S. typhimurium and an increase in the mice survival rate were observed. The P. freudenreichii component DHNA ingested by mice presenting a dextran sodium sulphate (DSS)-induced colitis led to a modulation of the microbiota, including a weaker drop in the Lactobacillus and Enterobacteriaceae intestinal populations caused by DSS (Okada et al. 2006b). DHNA also increased intestinal bifidobacteria population in mice suffering from non-steroidal anti-inflammatory drug (NSAID)-induced colitis (Okada et al. 2006a). Bifidobacterium is probably the most amply documented genus of the human microbiota studied for probiotic properties. Thus, stimulation of bifidobacteria growth by dairy propionibacteria therefore constitutes a key probiotic potential. Metabolic activity of P. freudenreichii has been described in the gastrointestinal tract of human microbiota-associated rats (Lan et al. 2007a). Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. Transcarboxylase is involved in propionic acid metabolism. Strain TL133 also increased the concentrations of acetate, propionate and butyrate in rat caecal contents (Lan et al. 2007a). In another study, mice fed P. acidipropionici CRL 1198 showed increased β-galactosidase activity and enhanced propionic acid levels in the caecum (Perez-Chaia and Zarate 2005). This strain also 14 F.J. Cousin et al. tends to reverse the hyperlipidemic effect of a high lipid diet (Perez-Chaia et al. 1995). This effect can be attributed to modulation of liver metabolism by absorbed propionic acid but can also be linked to the cholesterol-binding activity described above (Somkuti and Johnson 1990). Hypolipidemic effects of dairy propionibacteria should be confirmed in human studies. Many bacterial enzymes, including β-glucosidase, β-glucuronidase, azoreductase and urease, are involved in producing carcinogens within the gut. Many probiotic studies have monitored the activity of these faecal enzymes. A potential modulation of risk factors for carcinogenesis has been observed in animal models. In mice fed P. acidipropionici, β-glucuronidase activity was lower than in controls on a conventional diet (Perez-Chaia et al. 1999). With a red cooked meat supplement, the β-glucuronidase activity increased in the control mice. In the faeces of propionibacteria-supplemented mice, β-glucuronidase activity increased much less than in the control at the beginning of the meat diet and decreased thereafter. Propionibacteria supplementation prevented the increase in β-glucuronidase activity with the red meat diet. The authors also describe a slight reduction in azoreductase and nitroreductase activity (Perez-Chaia et al. 1999). Lan et al. (2008) report that P. freudenreichii TL133 increased induction of apoptosis in colonic mucosal crypts of human microbiota-associated rats treated with the carcinogen 1,2-dimethylhydrazine (DMH). The administration of propionibacteria alone did not increase the number of apoptotic cells in healthy colonic mucosa. This study demonstrates the ability of P. freudenreichii to favour apoptotic depletion of damaged cells at an early stage of malignant cell transformation in rats. As regards intestinal inflammation, in some studies, colonic infusion with P. acidipropionici (Michel et al. 2005) or oral supplementation with a milk whey culture of P. freudenreichii ET-3 (Uchida and Mogami 2005) reduced the severity of TNBS-induced colitis in rats. The healing of TNBS-induced colitis was also observed with oral administration of propionate (Uchida and Mogami 2005). In therapeutic and preventive studies, DHNA improved the survival rate and histological damage scores of mice with DSS-induced colitis (2006b). DHNA attenuated colonic inflammation not only by balancing intestinal bacterial ecosystem but also by suppressing lymphocyte infiltration (Okada et al. 2006b). Okada et al. (2006a) report that DHNA had an anti-inflammatory effect on NSAID-induced colitis in IL-10-knockout mice through increased numbers of Bifidobacteria and suppression of inflammatory cell infiltration. As regards immunomodulation, Perez-Chaia et al. (1995) observed an improvement in carbon clearance in mice fed with P. acidipropionici CRL 1198, indicating an enhanced phagocytic function of the reticuloendothelial system. Administration of this strain prior to S. typhimurium pathogen inoculation led to an increase in both anti-S. typhimurium IgA levels and numbers of cells producing the antibody (Alvarez et al. 1996). Moreover, P. acidipropionici enhanced the phagocytic activity of isolated mouse peritoneal macrophages (Perez-Chaia et al. 1995), which was higher with mice fed propionibacteria than in the controls. The orally administered P. acidipropionici showed this immunostimulating activitywith isolated cell wall but not with isolated peptidoglycan (Morata de Ambrosini et al. 1998). Immune system modulation by dairy propionibacteria may be related to the chemical composition of the cell walls, particular molecules protruding from the surface. Oral treatment of Dairy propionibacteria as probiotics 15 mice with P. freudenreichii subsp. shermanii JS in combination with L. rhamnosus GG increased T-cell and B-cell proliferation after stimulation with concanavalin A (T-cell mitogen), and lipopolysaccharide (B-cell mitogen), respectively (Kirjavainen et al. 1999). The authors suggest that these results may indicate that the splenic lymphocytes acquired a higher tolerance to the cytotoxic effects of the mitogens. They suggest that they are related to the capacity of dairy propionibacteria to bind this lectin in vitro, as cited above. Another study showed a higher T-cell proliferation of splenic lymphocytes with P. jensenii 702 inmice receiving soluble Mycobacterium tuberculosis antigens (Adams et al. 2005). The strain has been patented as an adjuvant for oral vaccines (Adams et al. 2003, 2008).

6 Probiotic potentialities of dairy propionibacteria in human

Many teams have studied the impact of dairy propionibacteria on the human microbiota. In several independent studies, ingestion of P. freudenreichii in the form of whey cultures, whether heat-inactivated (Kaneko 1999; Satomi et al. 1999) or not (Hojo et al. 2002), or as freeze-dried live bacteria forms (Bouglé et al. 1999; Roland et al. 1998), resulted in a higher faecal bifidobacteria population in human beings. Sarkar and Misra (1998, 2002) showed that a fermented milk containing B. bifidum, P. freudenreichii subsp. shermanii and either L. acidophilus or not led to a decline in coliforms and to an increase in bifidobacteria in the faeces of infants receiving the product. Whey cultures with P. freudenreichii ET-3 also triggered a decrease in Clostridium perfringens (Seki et al. 2004) and Bacteroides populations (Mitsuyama et al. 2007). In children suffering from intestinal dysbacteriosis, consumption of a milk containing P. freudenreichii subsp. shermanii and L. acidophilus restored of the microbiota, thus shortening the convalescence period during antibiotherapy (Sidorchuk and Bondarenko 1984). Hatakka et al. (2007) report that a cheese containing a mixture of probiotics (L. rhamnosus GG, L. rhamnosus LC705 and P. freudenreichii ssp. shermanii JS) reduced the risk of high yeast counts, especially Candida sp., in the mouth of elderly people. These authors also observed that probiotic intervention reduced the risk of hyposalivation and a dry mouth sensation and can therefore be considered beneficial to oral heath in general. In fact, the authors suggest that the absence of protective probiotics and increased hyposalivation might explain the enhanced Candida growth in the control group. A probiotic supplementation with L. rhamnosus GG, L. rhamnosus LC70, B. breve Bb99 and P. freudenreichii ssp. shermanii JS did not significantly reduce the frequency of new or aggravated symptoms caused by antibiotic treatment during H. pylori eradication (Myllyluoma et al. 2005), although this probiotic combination has exhibited promising anti-Helicobacter properties in vitro (see section above). However, taking total symptom severity into account, this study suggests improved tolerance of the anti-H. pylori treatment. The authors also show that the probiotic bacteria survived in the gastrointestinal tract despite the intensive antimicrobial therapy (Myllyluoma et al. 2005). A more recent study has confirmed that the same probiotic mixture counteracts the effects of antibiotic treatment against H. pylori (Myllyluoma et al. 2007a). In particular, reduced H. pylori-induced inflammation of the gastric mucosa was observed (Myllyluoma et 16 F.J. Cousin et al. al. 2007b). Altogether, these results strongly suggest that the tested probiotics, containing P. freudenreichii subsp. shermanii JS, may be helpful during the treatment of H. pylori infection. Hojo et al. (2002) report that a high faecal bifidobacteria level due to P. freudenreichii ET-3 supplementation was linked to an increased number of defecations in constipated female volunteers. Stool frequency was also significantly increased by administration of a P. freudenreichii culture in healthy human subjects (Kaneko 1999) and in elderly subjects (Seki et al. 2004). In another study, elderly subjects receiving a L. rhamnosus and P. freudenreichii-supplemented juice also exhibited an increase in defecation frequency (Ouwehand et al. 2002a). This probiotic property is interesting because constipation is a common problem in elderly subjects. Another clinical study reported a limited effect on segmental colonic motility, including slower transit in the left colon (Bouglé et al. 1999). The authors suggested that propionibacteria may regulate transit when this one is disturbed. The consumption of a juice supplemented with L. rhamnosus LC-705 and P. freudenreichii subsp. shermanii JS led to decreased faecal azoreductase activity in elderly subjects (Ouwehand et al. 2002a). The same probiotic combination was used in healthy men and led to a non-significant decrease in β-glucosidase activity of 10% and in urease activity of 13% (Hatakka et al. 2008). In irritable bowel syndrome patients, a probiotic mixture containing L. rhamnosus GG, L. rhamnosus LC-705, B. breve Bb99 and P. freudenreichii subsp. shermanii JS led to a decrease in β- glucuronidase activity in most people in the probiotic group (Kajander et al. 2007). A clinical trial investigated the effect of a probiotic preparation containing L. rhamnosus LC-705 and P. freudenreichii subsp. shermanii JS on levels of aflatoxin B1 in human faecal samples. Following probiotic administration, there was a significant reduction in the faecal level of aflatoxin B1 and this decrease continued during the follow-up period (El-Nezami et al. 2000b). Another study with the same probiotic supplementation in young men from Southern China found an increase in 7 urinary samples with negative aflatoxin B1-N -guanine and a decrease in the 7 concentration of urinary aflatoxin B1-N -guanine in the probiotic group (El-Nezami 7 et al. 2006). Aflatoxin B1-N -guanine is a marker for a biologically effective dose of aflatoxin. The probiotic supplementation thus reduced the organism’s exposure to this carcinogen. A commercial preparation of bifidogenic growth stimulator (BGS), which is produced by P. freudenreichii ET-3, led to an improvement in the clinical activity scores of ulcerative colitis patients (Mitsuyama et al. 2007; Suzuki et al. 2006). Patients also showed a decrease in the endoscopic index and an improvement in serum haemoglobin and albumin concentrations (Suzuki et al. 2006). An increase in all SCFA concentrations was measured after BGS ingestion but this was significant only for butyrate (Suzuki et al. 2006). The authors suggest that BGS restores a healthy microbial balance, thus favouring beneficial competitive interactions which may prevent or treat ulcerative colitis. Kekkonen et al. (2008b) report that a probiotic intervention with P. freudenreichii subsp. shermanii JS in healthy adults led to a reduction in the serum level of C- reactive protein (CRP) compared to a placebo control. CRP being a sensitive inflammation marker, this result confirms the anti-inflammatory potential of dairy Dairy propionibacteria as probiotics 17 propionibacteria. In a randomised, placebo-controlled, double-blind trial was performed in Helsinki on infants at high risk of allergy, a probiotic mixture containing L. rhamnosus GG, L. rhamnosus LC-705, B. breve Bb99 and P. freudenreichii subsp. shermanii JS was given daily for 6 months after birth and compared to a placebo (Kuitunen et al. 2009; Kukkonen et al. 2007, 2008). Two years after birth, less antibiotic prescription and fewer respiratory infections were reported in the supplemented infant group, whatever the mode of delivery (Kukkonen et al. 2008). In addition, less eczema and less atopic eczema were diagnosed in the treated group (Kukkonen et al. 2007). Five years following birth, significant differences were detected among caesarean-delivered children: less IgE- associated disease occurred, particularly eczema, and less IgE sensitisation was detected (Kuitunen et al. 2009). Thus, during the first stages of life, probiotic supplementation including propionibacteria seems to promote immune system maturation, preventing infections and allergies. In addition, such supplementation would further counteract disorders linked to caesarean delivery such as delayed colonisation of the gut by bifidobacteria and lactobacilli. Another randomised, placebo-controlled, double-blind trial tested the same probiotic mixture in infants with atopic eczema-dermatitis syndrome (AEDS) and suspected cow’s milk allergy (CMA). Soluble E-selectin and plasma IL-10 levels were higher after probiotic supplementation than after placebo treatment (Viljanen et al. 2005b) and faecal IgA levels tended to be higher in the probiotic group (Viljanen et al. 2005a). Another study showed that a mixture of P. freudenreichii subsp. shermanii JS, L. rhamnosus GG and LC-705 and B. breve Bbi99 increased IL-4 secretion and tended to stimulate IFN-γ secretion in PBMCs of infants with CMA (Pohjavuori et al. 2004). This may offer clinical benefits in the treatment of allergic diseases by immunologic means. Altogether, these clinical data indicate beneficial immunomodulation by a mixture of gram-positive bacteria including P. freudenreichii subsp. shermanii JS, with a reported anti-inflammatory effect of the latter. Further clinical data should pin down the role of dairy propionibacteria per se.

7 Production of other compounds and future trends

Many other properties of dairy propionibacteria can be regarded as beneficial (Hugenholtz et al. 2002). They secrete bacteriocins (Holo et al. 2002), anti-fungal compounds (Lind et al. 2007) and anti-viral compounds (Cutting et al. 1962; Furusawa et al. 1965, 1967; Ramanathan et al. 1965, 1966, 1968, 1973) and are therefore used as food preservatives. Propionate, being an SCFA, has been investigated for its health effects. In particular, preliminary in vitro investigations suggest that propionate has a role inducing apoptosis of gastric cancer cells (Matthews et al. 2007) and in preventing colon cancer cell colonisation (Emenaker and Basson 1998). In vivo, propionate increased secretion of intestinal mucus in rats (Shimotoyodome et al. 2000) and human colorectal calcium absorption (Trinidad et al. 1999). The suggested beneficial effects of propionate suggest similar properties for dairy propionibacteria, mediated by their metabolism end-products. As an example, induction of the satietogen peptide PYY by SCFAs having been reported (Cherbut et al. 1998), Ruijschop et al. (2008) tested a dairy product fermented by 18 F.J. Cousin et al. lactic acid bacteria and propionibacteria in a human study. After consumption of this product, subjects felt significantly fuller, were less hungry and had less desire to eat (Ruijschop et al. 2008). However, the authors report no effect on ad libitum food consumption. Also of health interest is the capacity of dairy propionibacteria to improve the nutritional quality of fermented products by synthesising vitamins, trehalose and conjugated linoleic acid (CLA). Dairy propionibacteria synthesise vitamin B8 (biotin), B9 (folic acid) and B12 (cobalamin) (Hugenholtz et al. 2002). Folic acid has been described as an agent for colorectal cancer prevention but this claim is controversial (Hubner and Houlston 2009). Trehalose has been observed to reduce the level of enterohaemorrhagic Escherichia coli O157:H7 in ruminants because this sugar can be used by commensal E. coli but not by the O157:H7 strain (de Vaux et al. 2002). The broad spectrum of biological effects reported for conjugated linoleic acids (Churruca et al. 2009; Wahle et al. 2004) includes the anticarcinogenic properties of rumenic acid, the cis-9,trans-11 stereoisomer of CLA (Lavillonniere et al. 2003; Lock et al. 2004). P. freudenreichii has been shown to convert linoleic acid to rumenic acid (McIntosh et al. 2009; Rainio et al. 2001, 2002; Vahvaselka and Laakso 2010; Wang et al. 2007) and the corresponding mechanisms have been identified (McIntosh et al. 2009). As the authors of that study suggest, rumenic acid formation in the human gut could promote health. The technological qualities of dairy propionibacteria constitute a key advantage for their uses as probiotics. They survive the technological stresses imposed during freeze-drying, spray-drying, reconstitution in milk, cheese making and storage at low temperatures. Dairy propionibacteria ferment a wide range of carbohydrate substrates. Such remarkable adaptability, robustness and versatility are consistent with the occurrence of propionibacteria in various niches and should allow their growth and/or viability in a variety of probiotic vectors. Most of the clinical studies have been conducted using mixtures of bacteria from different genera. The development of a pure culture of dairy propionibacteria in a food grade vector will make it possible to pin down their specific probiotic potential in human beings. However, synergistic effect between different probiotics should also be investigated in the different fields of beneficial activity. In addition, complex interactions between ingested probiotics and the complex gut microbiota should be taken into consideration. In this context, the ability of dairy propionibacteria to modulate this microbiota, beyond the propionibacteria population itself, is of particular interest. Probiotic properties “in general”, including bifidobacteria and lactobacilli, differ from a species to another and are straindependent. It is therefore necessary to screen a large number of dairy propionibacteria in order to select strains with the best potentialities for dedicated applications. In this context, such approach to study immunomodulation of a large set of diverse single propionibacteria both in vitro (PBMC) and in vivo (experimental colitis and infectious mice models) have already been initiated and developed (Deutsch/ Foligné et al., personal communication) and should fill some gaps soon. Besides classical screening methods, the first dairy Propionibacterium genome from a P. freudenreichii subsp. shermanii strain is expected to be available soon. Genomic data will allow new mechanistic investigations of its probiotic potential (Klaenhammer et al. 2005). Dairy propionibacteria as probiotics 19

8 Conclusion

In conclusion, although more experimental data are available concerning probiotic applications of bifidobacteria and lactobacilli, dairy propionibacteria also deserve attention. Indeed, there is now specific promising evidence from dairy propionibacteria regarding beneficial modulation of colon microbiota and carcinogenesis together with anti-inflammatory and immune properties. Future works should focus on the development of molecular tools and appropriate delivery vectors as well as on selecting the best strains. Progress on these aspects will allow specific clinical studies. These in turn should lead to a better understanding and exploitation of the beneficial effects of dairy propionibacteria in the different compartments of the gastrointestinal tract. Individual probiotics demonstrate unique, specific biological effects. Knowledge of the specific effects of each probiotic strain will allow the development of probiotic mixtures adapted to particular cases or pathologies. Selecting (mixtures of) probiotics for use in disease(s) treatment will be guided by improved knowledge on action mechanisms based on established experimental models from in vitro to clinic. Knowledge of probiotic mechanisms may allow us to select the strain(s) with the best possible expected biological outcome. However, bench-to-bedside clinical trials will remain necessary to validate the chosen strain selection strategy. It will also confirm the physiological effectiveness of the proposed mechanisms in, for example, a wider population of patients suffering immune disorders, such as Inflammatory Bowel Diseases (IBD) and Irritable Bowel Syndrome (IBS), as well as other gastrointestinal infection diseases, cancers and allergies.

Acknowledgements The authors thank Hariet Coleman for revising and correcting the English language. This work was financially supported by the Science Committee of Syndifrais and the Centre National Interprofessionnel de l’Économie Laitière (CNIEL). F.C. received a grant from CNIEL.

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